Valve and splitting system for multi-dimensional liquid analysis

ABSTRACT

A multi-dimensional liquid analysis system includes a flow splitter for separating mobile phase outflow from a first dimension liquid analysis system into first and second liquid split outlet flows. Volumetric flow rate control of the split outlet flows is provided by a flow control pump which withdraws one of the split outlet flows from the flow splitter at a controlled withdrawal flow rate to define the other split outlet flow rate as the difference between the outflow rate from the first dimension system and the withdrawal flow rate. In this manner, accurate and consistent flow division can be accomplished, which is particularly useful for multi-dimensional liquid analysis.

This application is a continuation of U.S. application Ser. No.14/996,784, filed Jan. 15, 2016 and entitled “Valve And Splitting SystemFor Multi-Dimensional Liquid Analysis,” which is a continuation of U.S.application Ser. No. 13/422,168, filed Mar. 16, 2012 and entitled “Valveand Splitting System for Multi-Dimensional Liquid Analysis,” whichclaims the benefit of U.S. Provisional Application No. 61/466,739, filedon Mar. 23, 2011 and entitled “Valve and Splitting System forTwo-Dimensional Liquid Chromatography,” the entire contents of eachbeing incorporated by reference herein.

FIELD OF THE INVENTION

The present invention relates to flow systems used in analyticalchemistry, and more particularly to a splitting system for splittingmobile phase flow in a multidimensional liquid chromatography apparatus.

BACKGROUND

Separation techniques such as high pressure liquid chromatography (HPLC)are commonly used in analytical chemistry. However, HPLC is limited bythe resolution which can be achieved using a single chromatographicseparation column. Attempts have been made to combine two or more liquidchromatographs into a hybrid instrument to achieve enhanced resolutionof more compounds than can be achieved in a single separation column. Assample complexity has increased over the years, a need has arisen forgreater resolving power than that achievable through the use of a singleHPLC column.

Some analytical instruments involve the combination of HPLC and massspectrometry for further identification of the sample. Typical massspectrometers, however, analyze a significantly lower flow rate than theflow rate typically passed through a HPLC separation column. Analystshave therefore attempted to operate such combined instrumentation byreducing the HPLC mobile phase flow rate to a less than optimum value sothat the outflow rate from the HPLC separation matches the liquid flowcapacity of the mass spectrometer. Such reduction in flow rate throughthe HPLC column tends to reduce the available chromatographicresolution. To avoid the reduction in HPLC resolution, flow splittershave been employed in a full-flow regime to split a portion of the flowfrom the outlet of the HPLC column or detector to the inlet of the massspectrometer, and the balance of the flow to another detector or towaste. Typical commercial flow splitters make use of resistive tubingelements to split the liquid flow into two or more distinct flowstreams. Example flow splitters are described in U.S. Pat. No.6,289,914, and European patent application Publication No. EP 495255A1.Resistive division of liquid flow is difficult to maintain at uniformlevels. Factors such as variable viscosity of the mobile phase,temperature, and any variations in the flow path during the analysis maycause the split ratio between the respective flow paths to change. Suchvariability becomes of particular concern when multiple dimension liquidchromatography is practiced.

One example chromatographic application where mobile phase splitting isdesirable is two-dimensional liquid chromatography (or LC*LC), whereinthe first dimension HPLC column effluent is introduced into a seconddimension HPLC column, with no portion of the first dimension separationnot being introduced into the second dimension column for subsequent“second dimension” separation. Those of ordinary skill in the art ofHPLC analysis understand the various techniques are known for injectinga sample into a chromatographic column. In many cases, a sample volumeis established in a multi-port valve, and thereafter injected into thechromatographic column by a fluid force generated by a pump. Samples maybe introduced into a flowing mobile phase stream.

Theoretically, it is desirable to have the entire volume of the firstdimension separation injected into the second dimension separationcolumn, though such an approach remains impractical as the rate of theeffluent from the first separation column is far too great to bedirectly injected into the second separation column. Traditionally,therefore, analysis of the “first dimension” separation has beenaccomplished by collection of the total volume of the effluent from thefirst separation column by fraction collection, and then re-injecting arepresentative sample of each fraction into the second dimensionseparation column.

In addition to flow rate mismatch, the developing chromatogram in thefirst dimension may contain increasing relative concentrations oforganic solvent. The increasing relative concentration of organicsolvent may be a result of the particular liquid chromatographicapproach, in which an organic solvent is injected into the separationcolumn after an aqueous mobile phase. As the relative concentration oforganic solvent increases in the first dimension separation, injectionof a fixed volume from the first dimension into the second dimensionchromatograph further increases the relative organic solventconcentration during the second dimension separation. Under someconditions, injecting large volumes of organic solvent into the seconddimension chromatograph is destructive to the second dimensionseparation. As the variation in organic solvent versus time occurs inthe first dimension separation, the flow rate exiting from standardresistive flow splitters disposed downstream from the first separationcolumn becomes unpredictable. Analysts therefore find it difficult toknow the actual flow rate of sample available for injection into thesecond dimension separation column. An understanding of the sample flowrate is critical to control the organic solvent concentration in thesecond dimension separation column, and to ensure that no portion of thefirst dimension chromatograph is unsampled in the second dimensionseparation. Typical resistive flow splitters are not capable ofproviding analysts with the necessary information to consistentlycontrol analysis in the second dimension. Because of the limitations ofstandard resistive flow splitters, LC*LC has not enjoyed wide usage inthe art.

SUMMARY OF THE INVENTION

A flow splitting system includes a T-style junction having an inlet forreceiving fluid exiting a first HPLC system separation column, a firstoutlet for permitting indirectly flow controlled outflow therefrom, thefirst outlet being fluidly coupled to a flow restricting device, and asecond outlet for permitting directly flow controlled outflow therefrom.The second outlet is fluidly coupled to a positive displacement pumpoperable in a negative displacement mode or a positive displacementmode. The flow restricting device creates a pressure in the junctionfrom 1 kilopascal to 10,000 kilopascals, and is an adjustable pressureregulator. The system further includes a positive displacement pump andvalve system wherein a 3-port shear type valve is used to connect a pumppiston and barrel to either the junction or to waste.

In some embodiments, the positive displacement pump is capable of beingdriven at a constant rate in the negative displacement mode and iscapable of withstanding pressure caused by the flow restricting deviceconnected to the first outlet of the junction.

In some embodiments, the positive displacement pump is driven at avariable rate in the negative displacement mode.

In some embodiments, the positive displacement pump is driven at anegative volume displacement rate which is less than the rate of solventinflow into the junction from the first dimension HPLC system.

In some embodiments, a 6-port sample injection valve for a seconddimension HPLC system is placed between the positive displacement pumpand the second outlet of the junction.

In some embodiments, a 10-port valve configured as a dual-loop injectorfor a second dimension HPLC system is placed between the positivedisplacement pump and the second outlet of the junction.

In some embodiments, the first outlet of the junction is connected to aninlet of a mass spectrometer.

In some embodiments, a draw time in which the positive displacement pumpdraws sample into the second dimension injection valve is equal to orslightly less or slightly more than an analysis time for a seconddimension separation.

In some embodiments, an injection loop volume in the second dimension6-port injection valve is either partially filled or fully filled duringa time of analysis of a previously injected sample and at a rate suchthat a complete representative sampling of all compounds exiting fromthe first dimension HPLC system enter a sample loop in the seconddimension injection valve.

In some embodiments, injection of the sample contained within the sampleloop of the second dimension injection valve takes place within one timestandard deviation of any analysis peak in a separation of the sampleperformed by the first dimension HPLC system separation column.

In some embodiments, the time that the second dimension injection valveremains in an inject position is less than 10 percent of the totalanalysis time of the second dimension HPLC system separation column.

A flow splitting system for liquid chromatography includes a T-stylejunction having an inlet for receiving fluid exiting a first HPLC systemflow-through detector, a first outlet for permitting indirectly flowcontrolled outflow therefrom, the first outlet being fluidly coupled toa flow restricting device, and a second outlet for permitting directlycontrolled outflow therefrom. The second outlet is fluidly coupled to apositive displacement pump operable in a negative displacement mode or apositive displacement mode. The flow restricting device creates apressure in the junction from 1 kilopascal to 10,000 kilopascals, or themaximum backpressure that said detector can withstand, and the flowrestricting device is an adjustable pressure regulator. The systemfurther includes a positive displacement pump and valve system wherein a3-port shear type valve is used to connect a pump piston and barrel toeither the junction or to waste.

In some embodiments, the positive displacement pump is capable of beingdriven at a constant rate in the negative displacement mode and iscapable of withstanding pressure caused by the flow restricting deviceconnected to the first outlet of the junction.

In some embodiments, the positive displacement pump is driven at anegative volume displacement rate which is less than the rate of solventinflow into the junction from a first dimension HPLC system.

In some embodiments, a 6-port sample injection valve for a seconddimension HPLC system fluidly coupled between the positive displacementpump and the second outlet of the junction.

In some embodiments, a 10-port injection valve configured as a dual-loopinjector for a second dimension HPLC system is fluidly coupled betweenthe positive displacement pump and the second outlet of the junction.

In some embodiments, the first outlet of the junction is connected to aninlet of a mass spectrometer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a schematic view of a system of the present invention;

FIG. 2 a schematic view of a system of the present invention;

FIG. 3 a schematic view of a system of the present invention;

FIG. 4 a schematic view of a system of the present invention;

FIG. 5 a schematic view of a system of the present invention; and

FIG. 6 a schematic view of a system of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

To effectuate consistent splitting of effluent flow from a firstdimension analysis column in a manner to preserve the first dimensionseparation resolution, a positive displacement pump, such as a syringepump, may be employed in a negative displacement mode to intake fluid ata specific rate from one outlet of a flow splitter. The resultant flowfrom a second outlet of the flow splitter is also therefore controlled.Such control dictates that the flow rate in both outlets of the split isknown.

A first schematic diagram of an arrangement of the present invention isprovided in FIG. 1. Analysis system 10 includes a first dimensionseparation system 12, and a second dimension separation system 14,wherein mobile phase is driven through a first dimension separationcolumn 16 by a first dimension pump 18. First dimension outflow 20 fromcolumn 16 may be delivered to a first dimension chromatographic detector22, or may first be split by a flow splitter. Flow rate into flowsplitter 24 is controlled by first dimension pump 18, which defines theflow rate of mobile phase through first dimension column 16. Flowsplitter 24 may comprise a T-style junction fitting having a first inletand first and second outlets, such as that available from Kinesis-USA asa “Micro-Splitter Valve 10-32/6-32 Port 55 Needle (EA)”. In thearrangement illustrated in FIG. 1, a first outlet 26 from flow splitter24 comprises a waste stream, while a second outlet 28 from flow splitter24 is at least intermittently fluidly coupled to flow control pump 30.In other embodiments, however, first outlet 26 may comprise a flowstream of known flow rate for delivery to a secondary analysis system,such as a mass spectrometer. System 10 is arranged such that firstoutlet 26 need only have sufficient flow restriction to avoidover-pressurization of pump 30 during the time that pump 30 controlsfluid flow through second outlet 28. In such a manner, control isexerted over both first and second outlets 26, 28 of flow splitter 24through control of the fill rate of pump 30 in a negative displacementmode. Consequently, no additional unswept volume is interposed betweenflow splitter 24 and any detection means (such as a mass spectrometer)fluidly coupled to first outlet 26.

Analysis system 10 performs chemical analysis of liquid sample pumpedinto first and second dimension columns 16, 34. For the purposes of thisinvention, first and second dimension “columns” may be construedbroadly, so as to include analysis modalities that do not necessarilyinvolve a column. For example, one or more of the dimensions may involveliquid chromatography, HPLC, preparative-scale liquid chromatography,supercritical fluid analysis, gel permeation chromatography, massspectrometry, other spectrometry or chromatography analysis, andcombinations thereof. In a particular application, the first and seconddimensions are each chromatographic columns for evaluating a liquidsample. In some embodiments, such liquid chromatography may be “highpressure liquid chromatography” or “high performance liquidchromatography” (HPLC), which is a common technique for performingchromatographic separations of solutions of compounds delivered to aninjection valve or “autosampler” by pump for injection into thechromatographic separation column. Liquids and liquid mixtures used totransport the compounds are referred to herein as the “mobile phase”.The “stationary phase” of liquid chromatography is typically the packingmaterials within the separation columns 16, 34.

As indicated above, flow control pump 30 may typically be a positivedisplacement pump having a displacement volume that is filled in anegative displacement mode, and discharged in a positive displacementmode. An example positive displacement pump is a syringe pump, in whicha plunger within a cylinder acts in a negative displacement mode to drawliquid into the cylinder through controlled withdrawal of the plungerthat creates a negative pressure within the cylinder to draw liquidtherein. Movement of the plunger in an opposite direction establishes a“positive displacement mode”, wherein a positive pressure is created inthe cylinder, so that the contents of the cylinder are dischargedtherefrom.

Flow control pump 30 may be placed downstream of any valve in secondoutlet stream 28, such that splitting of outflow 20 may be preciselycontrolled at known rates by controlling the rate at which pump 30 drawsfluid through second outlet stream 28. So long as the second outletstream 28 does not exceed the total flow rate at outlet flow 20, flowvolume versus time is known precisely in both first and second outletstreams 26, 28.

In the embodiment illustrated in FIG. 1, splitter 24 may be positionedbetween an outlet of first dimension column 16 and a second dimensioninjection valve 32. Depending upon the maximum volume of the firstdimension mobile phase from outlet 20 to be injected into seconddimension separation column 34, a fixed volume sample loop 36 of suchmaximum volume may be incorporated with the second dimension injectionvalve 32 in the form of a tube, channel, or other vessel capable oftemporarily containing the volume of sample mobile phase. Sample loop 36is more clearly illustrated in FIG. 2, with second dimension injectionvalve 32 being a 6-port injection valve, as is known in the art. In thisembodiment, sample loop 36 has a volume that is equal to or greater thanthe desired sample volume deliverable to the second dimension column 34.The programmed flow rate of pump 30 may be substantially equal to suchsample volume divided by the analysis time required of the seconddimension column 34. Such calculated withdrawal flow rate of flow stream42 ensures that a representative sample of all mobile phase passingthrough flow splitter 24 is delivered to second dimension column 34. Thefollowing sets forth a relationship for an example control scheme forpump 30 to establish an appropriate withdrawal flow rate from firstdimension outflow 20, and to therefore retain a sufficient seconddimension sample delivery flow rate to ensure complete chromatographicanalysis of mobile phase in second outlet stream 28:

F _(c) ≤V _(L)/(T _(2a) +T _(2e))

Wherein,

F_(c)=controlled flow rate at pump flow stream 42

V_(L)=volume of sample loop 36

T_(2a)=analysis time of second dimension column 34

T_(2e)=equilibration time of second dimension

The “equilibration time” of the second dimension is the time required to“flush” the second dimension column of an opposite-phase solvent. Forexample, certain HPLC analyses are performed by first passing an aqueousphase through the column, followed by an organic phase, with the samplebeing injected as appropriate into one or both of the aqueous/organicphases. The sample is eluted through the chromatographic column throughthe sequence of alternating aqueous/organic phases. Once the sample hasfinished eluting through the chromatographic column, it is desired thatthe column be “cleared” of any remaining aqueous/organic phase that isopposite to the initial mobile phase in the subsequent sample analysis.Therefore, in the example of a sample tested with first an aqueousphase, followed by an organic phase, such organic phase is preferably“flushed” from the column with blank aqueous phase (such as water) priorto initiating the subsequent sample sequence. This “flushing” time isthe “equilibration time” utilized in the above relationship.

An alternative embodiment is illustrated in FIG. 3, wherein firstdimension detector 22 is positioned downstream of flow splitter 24 influid communication with first outlet stream 26. A flow restrictor 50may be employed downstream of detector 22, or between splitter 24 andfirst dimension detector 22, in order to provide sufficient flowrestriction to enable pump 30 to operably control flow division atsplitter 24. Positioning flow restrictor 50 upstream of first dimensiondetector 22 may eliminate back pressure applied to detector 22 toimprove sampling accuracy. In any case, however, flow restrictor 50 isoptionally included, and is not necessary for the operation of thepresent invention.

A further embodiment of the invention is illustrated in FIGS. 4 and 5,wherein second dimension injection valve 32 is a 10-port valve, as isknown in the art. In such embodiment, two distinct flow paths may beestablished for use as a double loop injector. In a first flow path,sample may be directed through a first sample loop 36 a directly, whilein a second flow path, sample may be directed through a second sampleloop 36 b on valve 32. The difference in flow paths and the differencein resistance to flow is negated when using a flow splitter driven by apump 30 as described herein. First sample loop 36 a may be filled whilethe sample within second sample loop 36 b is analyzed in seconddimension column 34. In this case, pump 30 may be operated in a negativedisplacement mode to draw mobile phase from first dimension outletstream 20 sequentially into each of first and second sample loops 36 a,36 b. The draw rate through second outlet 28 may be such that the sampleloop volume being filled represents a volume suitable to be consumedover the entire time of the analysis and the equilibration of the seconddimension analysis (T_(2a)+T_(2e)). First and second sample loops 36 a,36 b may be alternately filled and injected to second dimension column34. An advantage of this technique is that each sample loop 36 a, 36 bis fully washed by the mobile phase of the second dimension over theentire time of analysis to eliminate carryover.

A waste discharge cycle of pump 30 is illustrated in FIG. 5. A valve 54may be employed to alternate between intake of mobile phase through flowstream 42 (as shown in FIGS. 2-4) and discharge to waste 56 throughwaste flow line 58 (as shown in FIGS. 5 and 6). In one embodiment,syringe pump 30 may be operated in a positive displacement mode todischarge its accumulated contents in a discharge time period (T_(d))that is less than one standard deviation in time for an analysis peakoccurring in the first dimension separation. In this manner, noparticular chromatographic analysis peak remains unsampled in the seconddimension chromatograph 34. In one embodiment, therefore, discharge timeT_(d) may be less than about 1 second.

Discharge from the pump 30 may occur only at intervals in which themobile phase substantially fills the displacement volume of pump 30. Forexample, first and second sample loops 36 a, 36 b may each be 20microliters in volume, while syringe pump 30 may have a displacementvolume of 5 ml. As a result, the displacement volume of pump 30 maybecome filled only after 125 injections into the second dimensionchromatograph 34.

A further embodiment is illustrated in FIG. 6, wherein system 110includes a flow splitter 124 disposed downstream from a first dimensioncolumn 116. First outlet stream 126 from flow splitter 124 is fluidlycoupled to an inlet of a secondary analysis device 170, such as a massspectrometer. A second outlet stream 128 from flow splitter 124 isfluidly coupled to a pump 130 to control the flow rate division at flowsplitter 124. As described above, pump 130 programmably removes solventfrom outflow stream 120 at a desired rate, such that the inlet flow rateto secondary analysis device 170 though first outlet stream 126 is equalto outlet flow rate 120 from first dimension column 116, less the flowrate being drawn into pump 130 through second outlet stream 128.

Fluid flow into the head of a liquid chromatographic column is notalways the total flow delivered to the inlet of the first dimension pump18 if the pump 18 is used to mix the mobile phase components, such as ingradient elution chromatography. Such an effect is caused by volumetricshrinkage of mixing, which results in the mixed mobile phase volumebeing less than the sum of the two individual liquid volumes. Toaccommodate such volumetric shrinkage of mixing, pump 30, 130 may beprogrammed according to the concentration of individual components ofthe mobile phase. An example relationship for such pump programming maybe as follows:

F _(i) =k*F _(o)

Wherein:

F_(i)=indirect flow rate

k=volumetric shrinkage factor

F_(o)=flow rate at outlet 120

Although an HPLC pump delivers a constant flow of each individualsolvent to a mixing point, the total flow rate from the mixing point maybe different from the sum of the individual liquid flows. As such, flowinto an HPLC column may be greater than the flow rate out of the HPLCcolumn when temperature is constant. The pump 30, 130 may therefore beadjusted to accommodate a difference between the inlet and outlet flowrates. Moreover, if the first dimension HPLC column 16, 116 is heated,the use of a pressurized, negative displacement flow splitter permitsall points of the second outlet stream 28, 128 to be pressurized suchthat boiling or outgassing of the mobile phase will not causeintermittent flow. In fact, when a first dimension column 16, 116 isheated, the use of negative displacement flow splitting may be the onlyeffective method for loading the sample loop of the injector withoutboiling of the solvent.

What is claimed is:
 1. A multi-dimensional liquid analysis system,comprising: a first separation system including a first separationcolumn, the first separation column configured to chromatographicallyseparate a sample within a liquid mobile phase and to provide a firstdimension outflow having a first outflow rate; a flow splitter fluidlycoupled to the first dimension outflow, the flow splitter configured tosplit the first dimension outflow into a first split outlet flow and asecond split outlet flow; a second separation system including a sampleloop and a second separation column, wherein the second separationsystem is configured such that the sample loop receives a sample volumefrom the second split outlet flow, and wherein the second separationcolumn is configured to chromatographically separate the sample volumefrom the second split outlet flow; and a flow controller fluidly coupledto and located downstream of one of the first split outlet flow and thesecond split outlet flow, the flow controller configured to control theflow rate of the one of the first split outlet flow and the second splitoutlet flow from the flow splitter at a controlled flow rate to obtain,in the sample volume from the second split outlet flow, a representativesampling of compounds in the first dimension outflow for separation inthe second separation system.
 2. The multi-dimensional liquid analysissystem of claim 1, further comprising: a flow restrictor restricting thefirst split outlet flow to create a fluid pressure at the flow splitterof 1-1000 kilopascals.
 3. The multi-dimensional liquid analysis systemof claim 1, wherein the flow controller is a positive displacement pumpconfigured to withdraw the one of the first split outlet flow and thesecond split outlet flow from the flow splitter at the controlled flowrate while operating in a negative displacement mode.
 4. Themulti-dimensional liquid analysis system of claim 1, wherein thecontrolled flow rate is controlled according to:${F_{c} \leq \frac{V_{s}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow,V_(s)≥V_(d), V_(d) is a volume of the second split outlet flow to beanalyzed by the second separation column, T_(2a) is an analysis time ofthe second separation column, and T_(2e) is an equilibration time of thesecond separation column.
 5. The multi-dimensional liquid analysissystem of claim 1, further comprising: a multiple-port injection valveconfigured to inject the sample volume from the second split outlet flowinto the second separation column.
 6. The multi-dimensional liquidanalysis system of claim 1, wherein to control the flow rate of the oneof the first split outlet flow and the second split outlet flow from theflow splitter, the flow controller is configured to produce a flow ratefor the second split outlet flow that is controlled according to:${F_{c} \leq \frac{V_{L}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow, V_(L)is a volume of the sample loop, T_(2a) is an analysis time of the secondseparation column, and T_(2e) is an equilibration time of the secondseparation column.
 7. A multi-dimensional liquid analysis system,comprising: a first separation system including a first separationcolumn, the first separation column configured to chromatographicallyseparate a sample within a liquid mobile phase and to provide a liquidmobile phase into a first dimension outflow having a first outflow rate;a flow splitter fluidly coupled to the first dimension outflow, the flowsplitter configured to split the first dimension outflow into a firstsplit outlet flow and a second split outlet flow; a second separationsystem including a multiple-port injection valve comprising a sampleloop, the second separation system also including a second separationcolumn, wherein the multiple-port injection valve is configured toreceive, at a port, the second split outlet flow such that the sampleloop receives a sample volume from the second split outlet flow, andwherein the second separation column is configured tochromatographically separate the sample volume from the second splitoutlet flow; and a flow controller fluidly coupled to and locateddownstream of one of the first split outlet flow and the second splitoutlet flow, the flow controller configured to control the one of thefirst split outlet flow and the second split outlet flow from the flowsplitter at a controlled flow rate to obtain, in the sample volume fromthe second split outlet flow, a representative sampling of compounds inthe first dimension outflow for separation in the second separationsystem.
 8. The multi-dimensional liquid analysis system of claim 7,further comprising: a flow restrictor restricting the first split outletlow to create a fluid pressure at the flow splitter of 1-1000kilopascals.
 9. The multi-dimensional liquid analysis system of claim 7,wherein the flow controller comprises a positive displacement pumpconfigured to withdraw the one of the first split outlet flow and thesecond split outlet flow from the flow splitter while operating in anegative displacement mode.
 10. The multi-dimensional liquid analysissystem of claim 7, wherein the controlled flow rate is controlledaccording to:${F_{c} \leq \frac{V_{s}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow,V_(s)≥V_(d), V_(d) is a volume of the second split outlet flow to beanalyzed by the second separation column, T_(2a) is an analysis time ofthe second separation column, and T_(2e) is an equilibration time of thesecond separation column.
 11. The multi-dimensional liquid analysissystem of claim 7, wherein the multiple-port injection valve isconfigured to inject the sample volume from the second split outlet flowinto the second separation column.
 12. The multi-dimensional liquidanalysis system of claim 7, wherein the controlled flow rate iscontrolled according to:${F_{c} \leq \frac{V_{L}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow, V_(L)is a volume of the sample loop, T_(2a) is an analysis time of the secondseparation column, and T_(2e) is an equilibration time of the secondseparation column.
 13. A method of multi-dimensional liquid analysis,comprising: chromatographically separating, using a first separationcolumn, a sample within a liquid mobile phase and providing, from thefirst separation column, a first dimension outflow having a firstoutflow rate; splitting, with a flow splitter having an inlet fluidlycoupled to the first dimension outflow, the first dimension outflow intoa first split outlet flow and a second split outlet flow; controlling,with a flow controller fluidly coupled to and located downstream of oneof the first split outlet flow and the second split outlet flow, the oneof the first split outlet flow and the second split outlet flow from theflow splitter at a controlled flow rate to obtain, in a sample volumefrom the second split outlet flow, a representative sampling ofcompounds in the first dimension outflow for separation in a secondseparation system; receiving, with a sample loop, the sample volume fromthe second split outlet flow; and chromatographically separating, usinga second separation column, the sample volume from the second splitoutlet flow.
 14. The method of claim 13, further comprising: modifying,without modifying the first outflow rate of the first dimension outflow,the flow rate of the one of the first split outlet flow and the secondsplit outlet flow from the flow splitter by reconfiguring the flowcontroller.
 15. The method of claim 13, further comprising: restricting,with a flow restrictor, the first split outlet flow to create a fluidpressure at the flow splitter of 1-1000 kilopascals.
 16. The method ofclaim 13, wherein the flow controller is a positive displacement pump,the method further comprising: configuring the flow controller in anegative displacement mode to withdraw the one of the first split outletflow and the second split outlet flow from the flow splitter at thecontrolled flow rate.
 17. The method of claim 13, further comprising:configuring a multiple-port injection valve to inject the sample volumefrom the second split outlet flow into the second separation column. 18.The method of claim 17, wherein the multiple-port injection valveincludes a port configured to receive the second split outlet flow. 19.The method of claim 13, further comprising: configuring the flowcontroller to produce a flow rate for the second split outlet flowaccording to:${F_{c} \leq \frac{V_{s}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow,V_(s)≥V_(d), V_(d) is a volume of the second split outlet flow to beanalyzed by the second separation column, T_(2a) is an analysis time ofthe second separation column, and T_(2e) is an equilibration time of thesecond separation column.
 20. The method of claim 13, furthercomprising: configuring the flow controller to produce a flow rate forthe second split outlet flow according to:${F_{c} \leq \frac{V_{L}}{\left( {T_{2\; a} + T_{2\; e}} \right)}},$wherein: F_(c) is the flow rate for the second split outlet flow, V_(L)is a volume of the sample loop, T_(2a) is an analysis time of the secondseparation column, and T_(2e) is an equilibration time of the secondseparation column.